Method of examining zinc-deficient taste disturbance by determining expression level of taste receptor genes

ABSTRACT

The present invention provides: a method for testing zinc deficiency dysgeusia, which is characterized in that it comprises correlating the expression levels of a gene encoding a gustatory receptor belonging to the THTR family and a gene encoding a gustatory receptor belonging to the T2R family obtained from a sample derived from the oral cavity of a subject, with a serum zinc level obtained from the sample of the subject; and a kit used for the aforementioned test.

TECHNICAL FIELD

The present invention relates to a method for testing zinc deficiency dysgeusia using the expression level of a taste substance-reactive composition (gustatory receptor) and a serum zinc level as indicators, and a kit for testing zinc deficiency dysgeusia.

BACKGROUND OF THE INVENTION

Gustatory sense is an important sense for life. Thus, abnormal gustatory sense gives a strong stress to patients' mentality. However, since such gustatory sense is a subjective sense, which can be recognized by only a person in question, it is difficult to objectively grasp the pathologic condition thereof. Hence, it is hard to say that an effective treatment is carried out for patients suffering from dysgeusia. Moreover, since such disorder of the sense of taste is not directly associated with the life-or-death matter of humans, it hardly becomes the object of interest for researchers. To date, dysgeusia has been tested by two main types of methods. One of them is called electrogustometry. This is a method, which comprises passing a weak current through the tongue and determining the disorder of the sense of taste based on the degree of the feeling obtained when electrical stimulation that is similar to taste is given. However, it is difficult for this method to detect abnormalities of individual qualities of tastes such as sweetness or bitterness. The other method is called a filter disk method. The filter disk method comprises placing a filter impregnated with each of taste substances in various concentrations on various sites of the tongue and hearing opinions from the subjects regarding the presence or absence of the taste. This method enables detection of the degree of feeling of 4 basic tastes such as sweetness, saltiness, sourness and bitterness, or the correctness of the feeling. However, this method is problematic in that good results cannot be obtained if subjects do not reach age when they understand the concept of this test, or if they do not have enough knowledge. Moreover, this method is also problematic when subjects do not know how to express the taste although they feel the taste. Thus, in a case where such subjects are infants and toddlers, or are elder people who are hearing-impaired, this test has almost no sense.

The aforementioned method enables detection of the presence or absence of dysgeusia, but it cannot clarify the cause of such dysgeusia.

By the way, in the case of mammals, the sense of taste is considered to be classified into 5 types of tastes such as sweetness, bitterness, delicious taste, sourness, and saltiness. Each quality of taste is considered to be transmitted via each different communication system. It is said that G protein-coupled receptors (GPCR) are involved in transmission of sweetness, bitterness, and delicious taste, and that ion channels are deeply involved in transmission of saltiness and sourness. However, the relationship between such receptors and taste substances has not yet been clarified. If such gustatory receptors (GPCR or channel proteins) selectively activate or inactivate towards chemical stimulation from the outside world, it is considered that cells on the tongue (taste cells) that express such receptors respond to such stimulation, and that information regarding a taste substance is transmitted to the central nerve.

As stated above, several gustatory receptors belong to the G protein-coupled receptors having a seven-transmembrane structure. In recent years, studies regarding G protein-coupled receptors for taste substances have vigorously been progressing. At current, T1R, T2R, and THTR families have been identified as gustatory receptors (Takeda et al. FEBS Lett. 520, 97-101, 2002) (National Publication (in Japan) of International Patent Application Nos. 2003-530098 and 2003-510037). However, the type of a substance used as a ligand (agonist) of such receptors, and in particular, of the THTR family, is still unknown.

Accordingly, it is desired to clarify such gustatory receptors, and to find a ligand acting as a taste substance. In addition, it is also desired to develop a novel method for testing dysgeusia, which does not depend on the personal view of a subject.

DISCLOSURE OF THE INVENTION

It is an object of the present invention to provide a method for detecting zinc deficiency dysgeusia and a kit for detecting dysgeusia.

As a result of intensive studies directed towards achieving the aforementioned object, the present inventor has established a method for testing zinc deficiency dysgeusia by making comparisons regarding the expression frequencies of gustatory receptor genes based on the causes of dysgeusia, thereby completing the present invention. That is to say, the present invention is as follows:

(1) A method for testing zinc deficiency dysgeusia, which is characterized in that it comprises correlating the expression levels of a gene encoding a gustatory receptor belonging to the THTR family and/or a gene encoding a gustatory receptor belonging to the T2R family obtained from a sample derived from the oral cavity of a subject, with a serum zinc level obtained from the sample of the subject. (2) The method according to (1) above, wherein the gustatory receptor belonging to the THTR family is at least one selected from the group consisting of THTR 1, 2, 3, 4, 5, 6, 7, 9, 11, 12, and 14. (3) The method according to (1) above, wherein the gustatory receptor belonging to the THTR family is a polypeptide described in (a) or (b) below: (a) a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22; or (b) a polypeptide having an amino acid sequence comprising a deletion, substitution, or addition of one or several amino acids with respect to the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and functioning as a gustatory receptor. (4) The method according to (1) above, wherein the gustatory receptor belonging to the THTR family is a polypeptide encoded by DNA described in (a) or (b) below: (a) DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21; or (b) DNA hybridizing with DNA having a nucleotide sequence complementary to the DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21 under stringent conditions, and encoding a polypeptide functioning as a gustatory receptor. (5) The method according to (1) above, wherein the gustatory receptor belonging to the T2R family is at least one selected from the group consisting of T2R 1, 3, 4, 5, 7, 8, 9, 10, 13, 14, and 16. (6) The method according to (1) above, wherein the gustatory receptor belonging to the T2R family is a polypeptide described in (a) or (b) below: (a) a polypeptide having the amino acid sequence as shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44; or (b) a polypeptide having an amino acid sequence comprising a deletion, substitution, or addition of one or several amino acids with respect to the amino acid sequence as shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44, and functioning as a gustatory receptor. (7) The method according to (1) above, wherein the gustatory receptor belonging to the T2R family is a polypeptide encoded by DNA described in (a) or (b) below: (a) DNA having the nucleotide sequence as shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43; or (b) DNA hybridizing with DNA having a nucleotide sequence complementary to the DNA having the nucleotide sequence as shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43 under stringent conditions, and encoding a polypeptide functioning as a gustatory receptor. (8) The method according to (1) above, wherein the sample derived from the oral cavity is a tongue tissue sample collected by a scratch method. (9) The method according to (1) above, wherein the expression level of a gustatory receptor gene is tested by amplification, and such amplification is carried out by amplifying the entire length of the gustatory receptor by RT-PCR. (10) A kit for testing zinc deficiency dysgeusia, which is characterized in that it comprises a primer designed from DNA described in (a) or (b) below: (a) DNA having the nucleotide sequence as shown in SEQ ID NOS: 45 to 88; or (b) DNA hybridizing with DNA having a nucleotide sequence that is complementary to the DNA having the nucleotide sequence as shown in SEQ ID NOS: 45 to 88 under stringent conditions.

According to the present invention, a method for testing zinc deficiency dysgeusia is provided. Differing from the conventional techniques, the method of the present invention does not rely on the feeling of human, and thus it is able to test dysgeusia more objectively. In addition, using the test method of the present invention, it is possible to make the diagnosis of zinc deficiency dysgeusia and the prognosis of such zinc deficiency dysgeusia case. Thus, the test method of the present invention is extremely useful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing the classification results of dysgeusia based on the causes thereof, and the results obtained by making comparisons regarding the expression frequencies of gustatory receptor genes based on the causes of dysgeusia.

FIG. 2 is a view showing the results obtained by making comparisons regarding the expression frequencies of gustatory receptor genes during the treatment of zinc deficiency dysgeusia cases.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be described in detail below. However, the following embodiments are provided to explain the present invention, and thus the present invention can be carried out in various embodiments unless it deviates from the gist thereof. All references and patent publications cited in the present specification are incorporated herein by reference in their entirety. In addition, the present specification includes the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 2005-376717, which is a priority document of the present application.

1. Summary

The present invention provides a method for testing dysgeusia, which is based on the correlation between a change in the expression level of multiple GPCR receptors that are considered to accept sweetness, bitterness, and delicious taste, and a serum zinc level. The method of the present invention is different from conventional methods for testing dysgeusia using the feeling of human as an indicator for determination. The present invention provides a method for testing dysgeusia, which comprises confirming the expression of multiple gustatory receptors by the RT-PCR method using a sample derived from the oral cavity and correlating the aforementioned expression level with a serum zinc level.

The present inventor has classified patients suffering from dysgeusia into those having zinc deficiency dysgeusia, idiopathic dysgeusia, influenza-induced dysgeusia, and drug-induced dysgeusia. The inventor has carried out a dysgeusia test regarding gustatory receptor genes that express in the tongue tissues of each patient. That is to say, the inventor detected by an RT-PCR method the expression level of 1-1 types of receptors (T2R 1, 3, 4, 5, 7, 8, 9, 10, 13, 14, and 16) that had already been reported as gustatory receptors and also the expression level of 11 types of receptors (THTR 1, 2, 3, 4, 5, 6, 7, 9, 11, 12, and 14) that had been considered to be candidates for gustatory receptors according to Takeda et al. (Takeda et al., FEBS Lett. 520, 97, 2002) in the tissues (human tongue tissues) of the aforementioned patients, and the inventor then analyzed to correlate such a gene expression level with a serum zinc level.

As a result, when compared with dysgeusia cases induced by other causes (e.g. idiopathic dysgeusia, influenza-induced dysgeusia, drug-induced dysgeusia, etc.), in the case of zinc deficiency dysgeusia, the expression frequency of a gustatory receptor gene on the tongue tissues tended to be low. In addition, in a case where the symptoms of zinc deficiency dysgeusia were not improved during the treatment thereof, the expression frequency of a gustatory receptor gene did not change. On the other hand, in a case where the symptoms of zinc deficiency dysgeusia were improved by continuous administration of a zinc preparation, the expression frequency of a gustatory receptor gene increased, and thus it tended to reflect the therapeutic effects.

Thus, the present invention enables testing of dysgeusia more objectively. Moreover, the present invention can be used in the diagnosis of zinc deficiency dysgeusia and it can also be used in the prognosis of zinc deficiency dysgeusia cases.

It is anticipated that a change in the expression level of a receptor that acts as a recipient of information brought by a taste substance be associated with dysgeusia. By testing such a change in the expression level of a receptor, it becomes possible to know the presence or absence of dysgeusia, the cause thereof, and a therapeutic target.

Furthermore, the present invention is useful in that a method of collecting tissues derived from the oral cavity is not carried out using a knife, but is carried out by almost painless noninvasive means (with a little burden on a subject).

2. Taste Substance-Reactive Composition (Gustatory Receptor)

The taste substance-reactive composition of the present invention (hereinafter referred to as a gustatory receptor at times) has a function to accept a taste substance and to transmit information obtained from the taste substance into the body. Among gustatory receptors, the gustatory receptor used in the present invention belongs to the THTR and T2R families included in GPCR. In particular, as such THTR family, THTR 1, 2, 3, 4, 5, 6, 7, 9, 11, 12, and 14 (amino acid sequences: SEQ ID NOS: 1 to 22 (only even numbers, namely, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, and 22)) can be used. As such T2R family, T2R 1, 3, 4, 5, 7, 8, 9, 10, 13, 14, and 16 (amino acid sequences: SEQ ID NOS: 23 to 44 (only even numbers, namely, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, and 44)) can be used. At that time, either gustatory receptors belonging to the aforementioned THTR family, or all of 22 gustatory receptors belonging to both the THTR and T2R families, may be used. Otherwise, several gustatory receptors may be used in combination, as appropriate. A list of the names of the aforementioned receptors used in the present invention, the nucleotide sequences thereof and the amino acid sequences thereof is shown in Table 1.

TABLE 1 SEQ ID NO. SEQ ID NO. Amino Amino Nucleotide acid Nucleotide acid Receptor name sequence sequence Receptor name sequence sequence THTR1/TAS2R38 1 2 T2R1/TAS2R1 23 24 THTR2/TAS2R47 3 4 T2R3/TAS2R3 25 26 THTR3/TAS2R45 5 6 T2R4/TAS2R4 27 28 THTR4-59 7 8 T2R5TAS2R5 29 30 THTR5/TAS2R40 9 10 T2R7/TAS2R7 31 32 THTR6/TAS2R39 11 12 T2R8/TAS2R8 33 34 THTR7 13 14 T2R9/TAS2R9 35 36 THTR9 15 16 T2R10/TAS2R10 37 38 THTR11/ 17 18 T2R13/TAS2R13 39 40 TAS2R48 THTR12/ 19 20 T2R14/TAS2R14 41 42 TAS2R49 THTR14/ 21 22 T2R16/TAS2R16 43 44 TAS2R44

Regarding receptor name in Table 1, TAS2RX (wherein X represents any given number) means a registered designation in GenBank.

In the present invention, among the aforementioned receptor genes, with regard to the THTR family, THTR11, THTR4 and THTR9 are preferably used, and with regard to the T2R family, T2R3, T2R8, T2R9, T2R10, T2R13 and T2R16 are preferably used.

In addition, the aforementioned GPCR-type gustatory receptors are associated with sweetness, bitterness, and delicious taste. These receptors also include those, which are estimated as gustatory receptors based on the homology of the amino acid sequences thereof because their taste substance acting as a ligand has not been identified.

Moreover, the gustatory receptor used in the present invention may comprise a mutation such as a deletion, substitution, or addition of multiple amino acids, and preferably one or several amino acids in polypeptides having the amino acid sequences as shown in the aforementioned SEQ ID NOS: 1 to 44 (only even numbers from SEQ ID NOS: 1 to 44), as long as it functions as a gustatory receptor.

A polynucleotide encoding an amino acid sequence comprising a deletion, insertion, substitution, or addition of one or several amino acids with respect to a certain amino acid sequence, can be prepared according to the site-directed mutagenesis described in Molecular Cloning, A Laboratory Manual 2nd ed. (Cold Spring Harbor Press (1989)); Current Protocols in Molecular Biology (John Wiley & Sons (1987-1997), and particularly, Section 8.1-8.5); Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-92; etc. Furthermore, a mutation can be introduced into a polynucleotide by known means such as the Kunkel method or the Gapped duplex method, using a mutation introduction kit that utilizes the site-directed mutagenesis, such as QuickChange™ Site-Directed Mutagenesis Kit (manufactured by Stratagene), GeneTailor™ Site-Directed Mutagenesis System (manufactured by Invitrogen), or TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, or the like; manufactured by Takara Bio).

Examples of a “function as a gustatory receptor” may include binding activity to bind to a taste substance and taste substance-mediated signal transduction action. The expression “a function as a gustatory receptor” is used to mean that the receptor has a function that is almost equivalent to that of a protein having an amino acid sequence as shown in each sequence number, for example. Accordingly, it is preferable that a protein, which comprises a mutation such as a deletion, substitution, or addition of multiple amino acids, and preferably one or several amino acids, with respect to the amino acid sequences (only even numbers from SEQ ID NOS: 1 to 44) as shown in SEQ ID NOS: 1 to 44 (only even numbers from SEQ ID NOS: 1 to 44), has activity equivalent to or greater than (approximately 0.5 to 1.5 times) the protein having the amino acid sequence as shown in each sequence number. However, quantitative elements such as the level of such activity or the molecular weight of the protein may be different.

Furthermore, the gustatory receptor used in the present invention also includes a partial peptide of the aforementioned receptor. An example of such a partial peptide used herein is a site of a receptor molecule, which is exposed to the outside of the cell membrane and has receptor-binding activity. Specifically, such a partial peptide of the receptor is a peptide comprising a portion that has been analyzed to be an extracellular region (hydrophilic site) as a result of hydropathy plot analysis. Further, a peptide comprising a hydrophobic site as a portion thereof can also be used. A peptide comprising individual domains can also be used. A partial peptide, which comprises multiple domains simultaneously, may also be used. In the present invention, the above partial peptide may comprise the aforementioned mutation such as a deletion, substitution, or addition in its amino acid sequence.

The type of DNA encoding the gustatory receptor used in the present invention is not particularly limited, as long as it encodes a polypeptide having the amino acid sequence as shown in any one of SEQ ID NOS: 1 to 44 (only even numbers from SEQ ID NOS: 1 to 44), a mutant thereof, or a portion thereof. Examples of such DNA include DNAs having the nucleotide sequences as shown in SEQ ID NOS: 1 to 44 (only odd numbers from SEQ ID NOS: 1 to 44 (1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, and 43)). In addition, DNA comprising a mutation such as a deletion, substitution, or addition of one or more nucleotides with respect to any one of the nucleotide sequences as shown in SEQ ID NOS: 1 to 44 (only odd numbers from SEQ ID NOS: 1 to 44) may also be included in the DNA of the present invention, as long as it functions as a gustatory receptor. Moreover, such DNA encoding the gustatory receptor polypeptide used in the present invention also includes DNA, which is capable of hybridizing with DNA having a nucleotide sequence that is complementary to the DNA having the nucleotide sequence as shown in any one of SEQ ID NOS: 1 to 44 (only odd numbers from SEQ ID NOS: 1 to 44) under stringent conditions, and which encodes a polypeptide functioning as a gustatory receptor. The term “stringent conditions” is used to mean conditions wherein a so-called specific hybrid is formed and a non-specific hybrid is not formed. Examples of such conditions are conditions wherein a complementary strand of DNA having high homology, that is, a complementary strand of DNA having homology of 60% or more, preferably 80% or more, more preferably 90% or more, and further more preferably 99% or more, hybridizes, and a complementary strand of DNA having homology lower than the above-described percentage does not hybridize. Specific examples of such stringent conditions may include conditions wherein a sodium concentration is set between 10 and 300 mM, and preferably between 20 and 100 mM, and wherein a temperature is set between 25° C. and 70° C., and preferably between 42° C. and 55° C.

In order to detect the amount of DNA encoding the aforementioned gustatory receptor polypeptide, the PCR method can be performed using each specific DNA.

The nucleotide sequences and amino acid sequences of the gustatory receptors used in the present invention are disclosed on the database of GenBank and the like. Using such genetic information, the primers of the present invention can be designed. Such primers should be designed such that only a receptor of interest can be amplified, when they are used in the PCR method. As DNA used to detect a change in the expression level of a gustatory receptor, the present invention includes, not only the aforementioned partial sequences (primers) capable of specifically amplifying the above gustatory receptor by the PCR method, but also DNA comprising a partial sequence (probe) specifically hybridizing with the above gustatory receptor, from among DNAs encoding gustatory receptor polypeptides. In order to examine the expression of a receptor gene with high sensitivity, it is preferable to amplify the receptor gene by the PCR method. As such a partial sequence, a sequence that amplifies the entire length of the THTR or T2R family may also be used. Moreover, such a partial sequence may be designed from each of DNA encoding the gustatory receptor polypeptide used in the present invention (e.g. SEQ ID NOS: 1 to 44 (only even numbers) and the mutants thereof) and DNA having a nucleotide sequence complementary thereto. Otherwise, such a partial sequence may also be designed from each of DNA encoding the gustatory receptor used in the present invention (e.g. SEQ ID NOS: 1 to 44 (only odd numbers) and the mutants thereof) and DNA having a nucleotide sequence complementary thereto. In the present invention, a primer preferably has a base length consisting of 18 to 27 nucleotides. In all of such cases, the partial sequence can be designed to amplify a full-length or a base length consisting of 300 to 1,000 nucleotides, preferably consisting of 500 to 900 nucleotides, and more preferably consisting of 700 to 800 nucleotides.

Furthermore, examples of the partial sequence of the present invention, and in particular, the primer of the present invention, may include primers used for amplifying the gustatory receptors shown in Table 2 set forth later, which are designed from DNA having the nucleotide sequence as shown in any one of SEQ ID NOS: 45 to 88, or DNA capable of hybridizing with DNA having a nucleotide sequence that is complementary to the DNA having the nucleotide sequence as shown in any one of SEQ ID NOS: 45 to 88 under stringent conditions. Stringent conditions are as described above.

The nucleotide sequences (SEQ ID NOS: 45, 47, . . . , 85, and 87) as shown in SEQ ID NOS: (2n+43) (wherein n represents an integer between 1 and 22) indicate the sequences consisting of 1^(st) to 18^(th)-26^(th) nucleotides with respect to the nucleotide sequences (SEQ ID NOS: 1, 3, . . . , 41, and 43) as shown in SEQ ID NOS: (2n−1) (wherein n represents an integer between 1 and 22). SEQ ID NOS: (2n+44) (n represents an integer between 1 and 22) (SEQ ID NOS: 46, 48, . . . , 86, and 88) indicate complementary sequences consisting of the last 18 to 26 nucleotides with respect to the nucleotide sequences as shown in SEQ ID NOS: (2n−1). Accordingly, SEQ ID NOS: (2n+43) and (2n+44) are pair primers, and such primers are able to amplify the entire length of DNA having the nucleotide sequence as shown in SEQ ID NO: (2n−1) by the PCR method.

A tag, a restriction enzyme recognition sequence, or the like, can appropriately be added to the 5′-terminal side of the primer of the present invention, as long as the above primer enables specific amplification of a receptor of interest.

DNA that is amplified with the primers of the present invention is DNA encoding the entire length or a part of a gustatory receptor polypeptide. From the view point of detection sensitivity and specificity in amplification, the length of DNA to be amplified is desirably the entire length of the gustatory receptor DNA. Further, it is also possible to add a necessary sequence to the 5′-terminal side of DNA used for amplification of the gustatory receptor used in the present invention. An example of such a sequence is a restriction enzyme recognition sequence.

The aforementioned primer or probe of the present invention can be produced according to known methods, or using a commercially available DNA synthesizer.

TABLE 2 Primer SEQ ID name Sequence NOS. THTR1/F atg ttg act cta act cgc atc 45 THTR1/R tca gca cag tgt ccg gga atc t 46 THTR2/F atg ata act ttt ctg ccc atc a 47 THTR2/R cta gaa gac aca caa tgc ccc tc 48 THTR3/F atg ata act ttt ctg ccc atc 49 THTR3/R tca gta cct cat ttg cca caa aac tg 50 THTR4/F atg gcc acc gaa ttg gac 51 THTR4/R cta caa agg taa agg gtt tgg tg 52 THTR5/F atg gca acg gtg aac aca gat g 53 THTR5/R tca cag agt ctg ccc ttt tag gt 54 THTR6/F atg cta ggg aga tgt ttt cct cc 55 THTR6/R tca cag agt cca ctc ttt tgg gt 56 THTR7/F atg cta ggg aga tgt ttt cct cc 57 THTR7/R tca cag agt ctg ccc ttt tag gt 58 THTR9/F atg ata act ttt cta ccc atc 59 THTR9/R cta tgg aga tga agt ctt ctc tcc 60 THTR11/F atg tta aag gac tca gaa caa g 61 THTR11/R2 tca gcg tgt cat ctg cca caa a 62 THTR12/F atg atg agt ttt cta cac att g 63 THTR12/R cta tgg agt tga ctg gtt ctg tcc 64 THTR14/F atg aca act ttt ata ccc atc 65 THTR14/R cta tgg aga tga agg ctt ctc tcc 66 T2R1/F atg cta gag tct cac ctc att atc 67 T2R1/R tca ctg aca gca ctt act gtg gag g 68 T2R3/F atg atg gga ctc acc gag ggg g 69 T2R3/R cta aga gaa aat ggg tcc ctt gg 70 T2R4/F atg ctt cgg tta ttc tat ttc 71 T2R4/R cta ttt ttt gaa aca aag aat c 72 T2R5/F atg ctg agc gct ggc cta gga ctg 73 T2R5/R tca tgg gcc cca gca tct ccg agc 74 T2R7/F atg gca gat aaa gtg cag act ac 75 T2R7/R tca gat ttg ttt atg ttg ttg ga 76 T2R8/F atg ttc agt cct gca gat aac 77 T2R8/R tca tat cat gca ggc aat ttt tc 78 T2R9/F atg cca agt gca ata gag gc 79 T2R9/R cta tgg aac aaa agg ctt tc 80 T2R10/F atg cta cgt gta gtg gaa ggc 81 T2R10/R cta tgt gac tct gag att ttt cc 82 T2R13/F atg gaa agt gcc ctg ccg ag 83 T2R13/R tca tcg ttt agc cca tac c 84 T2R14/F atg ctc tta cag gca atg gg 85 T2R14/R tca aga tga ttc tct aaa ttc 86 T2R16/F atg ata ccc atc caa ctc ac 87 T2R16/R cta gca ctt tcc ctt tag aat cc 88 3. Method of Collecting Oral Cavity-Derived Tissues

In the present invention, a change in the expression level of a gustatory receptor can be detected using RNA extracted from tissues derived from the oral cavity of a subject, such as a tongue tissue sample. As stated above, the present invention is characterized in that, in the test of dysgeusia, a knife is not used to collect tongue tissues, but the tongue tissues can be collected by almost painless noninvasive means (without bleeding).

Tongue tissues are collected by a scratch method. In this case, a sterile disposable microcentrifuge tube generally called “Eppendorf tube” can be used. Tongue tissues existing in the dorsum of tongue, fungiform papilla or foliate papilla of the tongue, or intraoral mucosal tissues existing in the backside of the cheek (Igaku Shoin, Keito Kango-gaku Koza, Senmon Kiso 1, Jintai no Kozo to Kinou [1], Kaibo-seiri-gaku (Systematic Nursing Science Seminar, Professional Base 1, Structure and Function of Human Body [1], Anatomical Physiology), written by Shigeaki Hinohara) were scratched 1 to 10 time, preferably 2 to 7 times, and more preferably 2 to 5 times, using the flange (edge) portion of a cap of the aforementioned tube, so as to obtain tissues from the tongue surface layer or the cheek. A site, from which such tissues are collected, is preferably around foliate papilla. After such tissues are collected from such a site by scratching, the site is preferably left for 2 or 3 days without collecting the tissues therefrom. The microcentrifuge tube is commercially available, and it can be purchased from companies such as Eppendorf or Assist. An unsterilized centrifugation tube may be purchased and may be then sterilized with an autoclave. Otherwise, a centrifugation tube, which has been sterilized, may be purchased. It is preferable that a centrifugation tube, which has been used once, be discarded, and that it be not reused for the purpose of collecting tongue tissues.

A tongue tissue sample is collected after the oral cavity has been fully washed with water or the like. Immediately after such a tissue sample is collected, 0.5 ml of an RNA extraction reagent such as TRIzol is added to the tube, followed by stirring and blending. Thereafter, the mixture can be conserved at −20° C. before use.

4. Method of Detecting Gustatory Receptor

Total RNA or mRNA is extracted from the tissues collected by the method described in the above “2. Method of collecting oral cavity-derived tissues” section. Thereafter, RT-PCR is performed. At the time, in order to extract RNA from the tissues, TRIzol (Invitrogen), Quick Prep Total RNA Extraction Kit (Amersham Biosciences), or RNeasy Kit (QIAGEN), can be used, for example. The obtained RNA is appropriately dissolved in DEPC-treated water (DEPC treated Water (Invitrogen), for example), and the concentration thereof is then measured. Thereafter, it can also be conserved at −80° C.

In addition, for a reverse transcription reaction, SuperScript III (Invitrogen) can be used as reverse transcriptase, and a random primer, an Oligo dT primer, a sequence-specific primer, or the like, can be used as a primer. Such a reverse transcription reaction may be carried out in accordance with a manual attached to the aforementioned enzyme.

In a PCR reaction, Ex Taq can be used as DNA polymerase. As primers used in the PCR reaction, those described in the aforementioned “1 Taste substance-reactive composition (gustatory receptor)” section (the primers as shown in Table 2, for example), can be used, for example. Conditions for PCR can be appropriately determined by persons skilled in the art in accordance with known methods. For example, PCR can be carried out under conditions wherein a reaction solution is treated at 94° C. for 3 minutes, thereafter, a cycle consisting of 94° C., 30 seconds, 59° C., 30 seconds, and 72° C., 1 minute, is repeated 35 time, thereafter, the resultant is treated at 72° C. for 7 minutes, and thereafter, the reaction is terminated at 4° C.

The method of carrying out RT-PCR of the present invention has two features. One feature is that 35 or more cycles are performed in the PCR. The other feature is that a receptor used as an amplification target is specifically and reliably amplified and detected.

With regard to the number of cycles applied in PCR, it has been known that, concerning the relationship between the number of cycles applied in PCR and the amount of a PCR product, the PCR product is first increased in an exponential manner, but that the PCR product reaches a plateau around 35 cycles (Shujunsha Co., Ltd., Bio Jikken Illustrated 3, Hontou nifueru PCR (Bio Experiment Illustrated 3, Real Amplification in PCR), written by Hiroki Nakayama). In the present invention, if PCR is performed in 35 or more cycles, the reaction product (PCR product) reaches a plateau regardless of the initial amount of a template, and thus it has a certain value (Shujunsha Co., Ltd., Bio Jikken Illustrated 3, Hontou ni fueru PCR (Bio Experiment Illustrated 3, Real Amplification in PCR), written by Hiroki Nakayama). Accordingly, it becomes more important to examine the presence or absence of the expression of such gustatory receptor RNA, than to examine the expression level of gustatory receptor RNA in the tissues. That is to say, in the case of the expression of the gustatory receptor, quantitative is not considered so much, but qualitative detection is mainly considered. Therefore, it can be interpreted that a PCR product, which is seen as a thin band at the stage of detection of the PCR reaction product (the stage of detecting the PCR product using 2100 Bioanalyzer manufactured by Agilent, for example), is hardly expressed. That is, a characteristic of the present invention is that the expression of a gustatory receptor is qualitatively examined using a reaction product wherein the amount of a PCR product has reached a plateau. As stated above, in the present invention, the cycle number applied in the PCR is 35 to 50 cycles, preferably 35 to 40 cycles, and more preferably 35 cycles.

Subsequently, reliable, specific amplification and detection of a receptor used as a target of amplification is essential when the presence or absence of the expression of a receptor family having high homology is detected by the PCR method. In order to confirm that an amplification product is a receptor as a target but is not another non-target receptor, several methods may be applied. For example, the analysis of the nucleotide sequence of the amplification product, amplification of the entire length of a receptor as a target by the PCR method, adoption of the base length to be amplified that differs for every receptor, etc., may be applied. At least a portion of a gene encoding such a gustatory receptor may be amplified. Among others, in terms of the easiness of primer design or prevention of amplification of similar nucleotide sequences, it is desired to amplify the entire length of a gene by the PCR method.

If the entire length of a gustatory receptor as a target is amplified by PCR, it is difficult to check the amplified PCR product itself. Therefore, the entire length of a gustatory receptor as a target should be amplified by the RT-PCR method, so as to confirm that normal amplification has been conducted and that the gustatory receptor as an amplification target has been certainly amplified, using the size of the amplified PCR product as an indicator. In addition, when the entire length is amplified, unless RNA that corresponds to the entire length of a gustatory receptor as a detection target exists in sample RNA, amplification is not successfully conducted. Thus, when the entire length is amplified, check is more reliably carried out than the case of amplifying at least a portion of a gustatory receptor gene. Moreover, when a trace amount of the RNA of a gustatory receptor that is not a detection target exists in a sample, amplification of such a trace amount of receptor gene becomes more difficult than amplification of a gustatory receptor as a detection target that is expressed in a large amount. Thus, amplification of the entire length of a gustatory receptor by the PCR method is more advantageous for the qualitative measurement of a gustatory receptor on the tongue, which is an object of the present invention. Accordingly, PCR primers are preferably designed such that they can amplify the coding region of a receptor as a whole (as mentioned above).

In order to measure the size or amount of a PCR product, a method comprising electrophoresing a PCR product by agarose gel electrophoresis, separating it depending on the size thereof, and measuring it depending on the thickness of a band, a method of measuring the molecular weight of a fragment contained in a PCR product using DNA LabChip (Agilent Technologies), or other methods, may be applied. Among others, 2100 Bioanalyzer manufactured by Agilent enables precise measurement of the size of a PCR product with high sensitivity, and thus it is effective for the test of the present invention. Using the thickness of the band of the thus obtained PCR product and/or the number thereof as an indicator, in the present invention, the presence or absence of dysgeusia is examined based on the fact that a gustatory receptor is not expressed. That is to say, when the expression of 10 different receptors is examined among multiple THTR receptors and T2R receptors, for example, if a certain number of thick bands of PCR products appear, or if a certain number of thick bands do not appear, it is determined that dysgeusia, glossalgia, or a state of stress has occurred. Since PCR is conducted until the PCR reaction product reaches a plateau in the present invention, it is considered that almost no receptors are expressed in the case of a PCR product that is seen as a thin band. Accordingly, such a thin band is not counted as a number of bands appeared.

The term “thin band” is used to mean a band having a peak area value (peak area) of 10 or smaller. In the present invention, 2100 Bioanalyzer manufactured by Agilent is used to measure the base length of a PCR product and the amount of a gene product amplified. 2100 Bioanalyzer is a device for electrophoresing a gene product such as a product amplified by PCR or the like and measuring the amount and base length thereof. This measurement device is used to obtain a base length (size) based on the time required until a gene product electrophoresing in a capillary due to electrical power passes through a detector and to indicate the size of a peak area indicating the strength of a signal detected by the detector as the thickness of a band. 2100 Bioanalyzer indicates the size of the peak area of a gene product signal as a pseudo-electropherogram of the expression pattern of a gustatory receptor gene. When the value of a peak area recorded during the measurement of such pseudo-electropherogram is 20 or greater, it is shown as a thick clear band. Thus, such a band is expressed as a “thick band” in the present invention, and it is determined that a gustatory receptor gene is expressed in tongue epithelial tissues. On the other hand, when such a peak area value is 5 or smaller, it is expressed as an extremely thin band, and it shows that almost no gustatory receptor genes are expressed in the present invention. When a value between 5 and 10 is obtained, the expression “only a low level of expression” is used. However, in this case, since gustatory receptor genes are expressed but a small number of receptors are expressed in tissues, it is assumed that such receptors do not function as gustatory receptors. In the case of a small peak that is not recognized by analysis software constituting 2100 Bioanalyzer, it is determined that no gustatory receptor genes are expressed. When this case is used for a mass examination of gustatory sense, if a peak area value is 10 or smaller, it may be determined that “no gustatory receptor genes are expressed (although there is gene expression, it does not have the function of feeling taste).”

5. Relationship Between Serum Zinc Level and Gene Expression Level

The present invention relates to a method for testing zinc deficiency dysgeusia. Accordingly, in the present invention, the expression level of the aforementioned gustatory receptor gene is measured, and at the same time, the serum zinc level of a patient is also measured. Thus, whether or not the patient suffers from dysgeusia can be determined by correlating the gene expression level with the serum zinc level.

Such a serum zinc level can be measured from the serum of the patient by a known method (e.g. an atomic absorption method). The normal level of zinc in serum is 65 to 110 μg/100 ml (http://www.mbcl.co.jp/compendium/main.asp?field=01&m class=09&s_class=0015; please refer to a practical guide of characteristics to be tested, Mitsubishi Chemical BCL).

It is to be noted that the normal level of zinc in serum is found to be 64 to 111 μg/100 ml by Mitsubishi Chemical BCL. An excessive level of zinc in serum hardly occurs in clinical sites. In contrast, there are far more pathologic conditions associated with a decrease in the zinc level in serum, and such pathologic conditions are considered to be serious. Zinc deficiency provokes various types of disorders such as growth and development disorder, gonadal failure, cutaneous lesion, or dysgeusia/dysosmia. Further, the relationship between serum zinc deficiency and dysgeusia has previously been well known. In accordance with the practical guide, as clinical significance, zinc is a typical, essential, trace metal existing in serum. The aforementioned practical guide describes that such serum zinc deficiency causes dermatitis or dysgeusia, and that the measurement of such a serum zinc level is important for the diagnosis of zinc deficiency disorders associated with central venous nutrition/enteral feeding.

Hence, in the present invention, if such a serum zinc level is low and if the majority of an electrophoretic band exhibiting the expression of a gustatory receptor gene on the tongue is not observed, the patient can be determined to have zinc deficiency dysgeusia. In a case where a serum zinc level is low and where a thin band is observed in the electrophoretogram of the present case although the thickness of the band of a PCR product is unbalanced, it can be determined that the patient is likely to have zinc deficiency dysgeusia.

6. Kit

The primer of the present invention can be provided as a kit for testing zinc deficiency dysgeusia. The test kit of the present invention (hereinafter referred to as “the present kit”) comprises components useful for carrying out the test method of the present invention, as well as a primer. Examples of such components may include the following (a) to (c):

(a) Instruments or Reagents Used when Total RNA is Extracted from Tissues

TRIzol solution, 1-mL disposable syringe with needle (e.g. top, disposable top plastic syringe 25 G×1″ R.B.), isopropanol, 70% ethanol for rinsing, and RNase free DEPC treated Water

The reagents and instruments described in (a) above are used as follows. 0.5 to 1 ml (preferably 0.5 ml) of a TRIzol solution per sample is placed in a tube that contains an oral sample, and the tube is then intensively stirred, so as to blend the oral sample attached to the cap of the tube with TRIzol. Thereafter, using the syringe, the oral sample in TRIzol is strongly sucked and discharged approximately 20 to 30 times in the tube, so as to facilitate extraction of RNA from the sample. Isopropanol is used in an amount that is half of the used TRIzol, so as to precipitate RNA. A tube used for such RNA precipitation is preferably RNase free. 70% ethanol for rinsing is used to rinse total RNA precipitated with isopropanol. RNase free DEPC treated Water is used to dissolve the obtained total RNA.

(b) Instruments and Reagents Used for Reverse Transcription of Total RNA

Reaction tube (DNase RNase free tube), dNTP, Random primer mixture, reverse transcription buffer, RNase Inhibitor, DTT, reverse transcriptase (preferably, SuperScript III (Invitrogen)), and RNase H

(c) Instruments and Reagents Used to Carry Out PCR Reaction

Reaction tube (DNase RNase free tube), PCR reaction buffer, dNTP solution, DNase-RNase free water, DNA polymerase (preferably, Ex Taq, Takara)

Examples of the aforementioned buffer may include a phosphate buffer and a Tris-HCl buffer (pH 4 to 10). Such buffer may comprise a surfactant such as SDS. Moreover, the present kit may also comprise a microcentrifuge tube for collecting an oral sample by the scratch method, a column for extracting RNA from the sample, a buffer, a surfactant, etc.

Using the present kit, the method for testing dysgeusia, method for testing glossalgia, and method for testing stress of the present invention can be easily carried out.

The present invention will be more specifically described in the following examples. However, the following examples are not intended to limit the technical scope of the present invention.

Example 1 Amplification of Gustatory Receptor Gene

(1) Method of Collecting Tissues

The left and right foliate papillae of a subject were collected by scratching 2 or 3 times with the flange (edge) portion of the cap of an Eppendorf tube.

Immediately after the aforementioned collection of tissues, 0.5 ml of TRIzol of Invitrogen was added to such tissues, and they were fully stirred and blended. Thereafter, the obtained mixture was conserved at −20° C. before use.

(2) RNA Extraction Method

Total RNA was extracted from the collected tissues. Extraction was conducted in accordance with a manual included with TRIzol. The obtained total RNA was dissolved in 10 μl of DEPC treated water (Invitrogen), and the concentration of the obtained total RNA was then measured, followed by conservation at −80° C.

(3) RT-PCR Method

0.65 μg of the obtained total RNA and a random primer (Invitrogen) were subjected to a reverse transcription reaction using Super Script III (Invitrogen). 0.5 μl of the obtained reaction solution was used as a template, and PCR was carried out in 50 μl of a reaction system using Ex Taq (Takara).

With regard to the THTR family genes, the PCR was performed on THTR11, THTR4 and THTR9, and with regard to the T2R family genes, the PCR was performed on T2R3, T2R8, T2R9, T2R10, T2R13 and T2R16. The used primers are shown in Table 2. Both the reverse transcription reaction and the PCR reaction were carried out in accordance with a manual included therewith. For PCR, GeneAmp PCR System 9700 manufactured by Applied Biosystems was used. PCR conditions consisted of a reaction at 94° C. for 3 minutes, 35 cycles of 94° C.-30 seconds, 59° C.-30 seconds, and 72° C.-1 minute, and a treatment at 72° C. for 7 minutes. Thereafter, the reaction product was then conserved at 4° C., until the measurement was carried out using 2100 Bioanalyzer of Agilent.

(4) Examination of PCR Product

The PCR product obtained under the aforementioned conditions was examined using 2100 Bioanalyzer of Agilent.

(5) Results

(5-1) Comparisons Regarding Expression Frequencies of Gustatory Receptor Genes Based on the Causes of Dysgeusia

Patients suffering from dysgeusia were classified into those having zinc deficiency dysgeusia, idiopathic dysgeusia, influenza-induced dysgeusia, and drug-induced dysgeusia (FIG. 1).

The age distribution and sex of the subjects are as shown in Table 3.

TABLE 3 Age distribution Sex 21-30 1 subject Male Female 31-40 3 subjects 41-50 3 subjects 7 subjects 16 subjects 51-60 5 subjects 61-70 7 subjects 71-80 4 subjects

The type of dysgeusia and the number of cases are as follows.

Zinc deficiency dysgeusia: 9 cases (39%)

Idiopathic dysgeusia: 7 cases (31%)

Influenza-induced dysgeusia: 3 cases (13%)

Drug-induced dysgeusia: 4 cases (17%)

Each of the aforementioned types of dysgeusia was determined as follows. It is to be noted that the type of dysgeusia was determined by a filter disk method.

Zinc deficiency dysgeusia: A type of dysgeusia determined by the filter disk method, which exhibits a low serum zinc level.

Drug-induced dysgeusia: A type of dysgeusia determined by the filter disk method, to the subject of which a drug causing dysgeusia (e.g. an antihypertensive agent exhibiting antagonism to serum calcium) has been administered.

Influenza-induced dysgeusia: A type of dysgeusia determined by the filter disk method, the subject of which has been infected with influenza.

Idiopathic dysgeusia: A type of dysgeusia determined by the filter disk method, which is not induced by the aforementioned causes.

RT-PCT was carried out regarding a gustatory receptor gene expressing in the tongue tissues of each patient. Based on the results of the electrophoresis, whether or not a clear band of the PCR product was expressed was analyzed. When only a thin band (a band like a line traced weakly with a pencil with hard lead of 3H or 4H, for example) was seen by visual observation, it was determined that the gustatory receptor gene was hardly expressed. The expression frequency (%) of thick, clear bands (bands like lines traced strongly with a pencil with soft lead of 2B or 3B, for example) is as shown in FIG. 1.

In FIG. 1, the four parts of the bar graph used for individual receptor genes or receptor candidate genes indicate the causes of dysgeusia, namely, zinc deficiency dysgeusia, idiopathic dysgeusia, influenza-induced dysgeusia, and drug-induced dysgeusia, from the left.

In FIG. 1, the THTR4 gustatory receptor candidate gene was not expressed in all the 9 cases of zinc deficiency dysgeusia patients, whereas the T2R3 gustatory receptor gene was expressed in 3 out of the 9 cases. FIG. 1 shows that, particularly in the zinc deficiency dysgeusia cases, the expression frequency of a gustatory receptor gene in the tongue tissues is likely to be lower than in other types of dysgeusia cases. These results demonstrated that the method of the present invention can be used in the diagnosis of zinc deficiency dysgeusia.

(5-2) Comparisons Regarding Expression Frequencies of Gustatory Receptor Genes During Treatment of Zinc Deficiency Dysgeusia Cases

Patients suffering from zinc deficiency dysgeusia were treated by administration of a zinc preparation. Every 5 weeks, a test of gustatory sense was carried out by a filter disk method, and the expression of a gustatory receptor gene in tongue tissues was analyzed.

The results are shown in FIG. 2. FIG. 2 is a view showing the results obtained by comparing the expression frequencies of gustatory receptor genes during the treatment of the zinc deficiency dysgeusia cases (the results of electrophoresis). The number in the parentheses indicates a zinc level in serum.

The electrophoretic bands indicate the expression conditions of a DNA size marker, 3 types of gustatory receptor gene THTR family, THTR11, THTR4 and THTR9, and 6 types of T2R family, T2R3, T2R8, T2R9, T2R10, T2R13 and T2R16, from the left of the figure. The rightmost band indicates the expression of β-actin selected as a gene for checking a reverse transcription reaction.

As shown in the upper case of FIG. 2, in one case where dysgeusia was not improved, the expression frequency of a gustatory receptor gene was not changed. In contrast, in four cases where dysgeusia was improved, as shown in the lower case of FIG. 2, the expression frequency of a gustatory receptor gene was increased as a zinc preparation was continuously administered, and thus it demonstrated that the therapeutic effects were reflected.

Accordingly, it was demonstrated that the method of the present invention can be used in the prognosis of zinc deficiency dysgeusia cases.

[Sequence Listing Free Text]

SEQ ID NOS: 45 to 88: primers 

1. A method for testing zinc deficiency dysgeusia, comprising: testing a sample from the oral cavity of a subject to determine the expression level of at least one gene encoding a gustatory receptor belonging to the THTR family and/or at least one gene encoding a gustatory receptor belonging to the T2R family; testing a serum sample of the subject to determine the serum zinc level; and correlating the gene expression levels of said gene or genes with the serum zinc level.
 2. The method according to claim 1, wherein the gustatory receptor belonging to the THTR family is selected from the group consisting of THTR 1, 2, 3, 4, 5, 6, 7, 9, 11, 12, and
 14. 3. The method according to claim 1, wherein the gustatory receptor belonging to the THTR family is a polypeptide described in (a) or (b) below: (a) a polypeptide having the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22; or (b) a polypeptide having an amino acid sequence comprising a deletion, substitution, or addition of one or several amino acids with respect to the amino acid sequence as shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22, and functioning as a gustatory receptor.
 4. The method according to claim 1, wherein the gustatory receptor belonging to the THTR family is a polypeptide encoded by DNA described in (a) or (b) below: (a) DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21; or (b) DNA hybridizing with DNA having the nucleotide sequence fully complementary to the DNA having the nucleotide sequence as shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21 under stringent conditions, and encoding a polypeptide functioning as a gustatory receptor.
 5. The method according to claim 1, wherein the gustatory receptor belonging to the T2R family is selected from the group consisting of T2R 1, 3, 4, 5, 7, 8, 9, 10, 13, 14, and
 16. 6. The method according to claim 1, wherein the gustatory receptor belonging to the T2R family is a polypeptide described in (a) or (b) below: (a) a polypeptide having the amino acid sequence as shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44; or (b) a polypeptide having an amino acid sequence comprising a deletion, substitution, or addition of one or several amino acids with respect to the amino acid sequence as shown in SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, or 44, and functioning as a gustatory receptor.
 7. The method according to claim 1, wherein the gustatory receptor belonging to the T2R family is a polypeptide encoded by DNA described in (a) or (b) below: (a) DNA having the nucleotide sequence as shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43; or (b) DNA hybridizing with DNA having the nucleotide sequence fully complementary to the DNA having the nucleotide sequence as shown in SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, or 43 under stringent conditions, and encoding a polypeptide functioning as a gustatory receptor.
 8. The method according to claim 1, wherein the sample derived from the oral cavity is a tongue tissue sample collected by a scratch method.
 9. The method according to claim 1, wherein the expression level of the at least one gustatory receptor gene is tested by amplifying the entire length of a gustatory receptor mRNA by RT-PCR. 